BRAF inhibitor (BRAFi) has emerged as one of the key treatments for BRAFV600E mutation which accounts for 60% of cutaneous melanomas. While the response rate is high and responses can be rapid, relapse is inevitable and often occurs within 6 months. The addition of MEK inhibitor (MEKi) could increase the duration of response from 6 to 9 months and increase one year survival to 40%. However, relapse invariably occurs and there is no effective salvage treatment available. Another approach is the use of immunotherapy with checkpoint inhibitors. Ipilimumab (anti-CTLA 4 antibody) and anti-programmed death 1 (anti-PD1) antibody (Nivolumab and Pembrolizumab) showed an increase in duration of response (99 weeks) and increased overall survival (63% one year survival rate). However, the time to response is often long for these agents, and when rapid disease progression occurs, especially after BRAF failure, patients may not be able to complete the planned treatment course, as has been reported for ipilimumab. Currently, there is a paucity of published data on the antitumor activity of anti-PD1 and anti-PD-L1 after BRAFi failure or BRAFi and MEKi failure patients. In this application, we plan to target to treat this group of patients which has rapid progressive disease and extremely poor prognosis. Our preliminary data showed that these resistant cells are very sensitive to arginine deprivation and pegylated arginine deiminase (ADI-PEG20) which degrades arginine to citrulline produced >80% decrease in these resistant tumors in vivo. We plan to investigate the underlying mechanism(s) and translate this into clinical trial. Our previou work and others have shown that certain melanomas do not express argininosuccinate synthetase (ASS), a key enzyme in the urea cycle, to synthesize arginine. These tumors depend on exogenous arginine to survive. ADI-PEG20 inhibits tumor growth with minimal toxicity since normal cells can take up citrulline to make arginine. However, we have found that autophagy does occur which can blunt the antitumor effect and re-expression of ASS can develop. These two mechanisms can account for drug resistance seen with this treatment. Importantly, we have found that cMyc is a positive regulator for re- expression of ASS. Interestingly, we have found that both BRAF resistant (BR) and dual resistant [resistant to both BRAFi and MEKi (BMR)] cells cannot readily undergo autophagy due to decrease in AMPK1-? and Atg5. This makes these resistant cells exquisitely sensitive to ADI-PEG20 treatment. In addition, both BR and BMR cells are unable to re-express ASS gene due to decrease in cMyc, and hence they cannot evade the apoptotic effect by ADI-PEG20 treatment. We plan to investigate how cMyc, AMPK1-?, and Atg5 are down regulated. Importantly, we plan to investigate the clinical relevance of our laboratory findings by examining tumor samples obtained from BR and BRM patients for AMPK1-?, Atg5 and ASS expression and for ADI-PEG20 sensitivity using tumor explant and primary culture as outlined in aim 2. Clinically, BR and BMR patients usually progress very rapidly and ADI-PEG20 alone may not be sufficient to control the tumor. In aim 3, we plan to investigate which combination with ADI-PEG20 yields the best antitumor efficacy. Based on our preliminary data, we plan to combine ADI-PEG20 with MEKi in BR tumors and ADI-PEG20 with Hsp-90 inhibitors in BMR tumors. Our ultimate goal is to apply this combination to treat this group of extremely poor prognosis patients. Immune checkpoint inhibitors have become a major component for the treatment of melanoma, and the effect of ADI- PEG20 on PD-1 and PD-L1 expression has not been explored. Our preliminary data showed that ADI-PEG20 can upregulate PD-L1 in BR and BMR cells, which may potentiate the antitumor effect of anti-PD-1 and anti- PD-L1 antibody. We plan to examine tumor explants, BR, and BMR cells for PD-1/PD-L1 expression before and after ADI-PEG20 treatment. Furthermore, we will also examine PD-1 expression from lymphocytes in these patients before and after treatment with ADI-PEG20. Thus, future combinatorial treatment using both agents can be rationally formulated to treat ASS (-) melanoma patients.